1) Field of the Invention
This invention relates to a new class of methine and polymethine dyes bearing one or two cyano groups on the alpha carbon atom relative to the nucleus of the dye compounds. The cyano group replacing the hydrogen on the alpha carbon atom next to the nucleus, adds to the photochemical stability of the dyes, increases its fluorescence efficiency and renders other physicochemical stability properties which are very important in the labeling of cells and other biological substrates. In one aspect of this invention, certain of the dyes, while forming non-fluorescent solutions in buffer or other solvents, fluoresce intensely when entering a cell.
A methine and polymethine dye consists of two nucleuses, one of which is positively charged, connected by an olefinic or polyolefinic group conjugated with the other nucleus, thau allowing the two nucleuses to share a resonance mesomeric effect. The group that connects the two nucleuses can also be cyclic, unsaturated, polar or non-polar and should satisfy the conjugation requirements of the dye.
The novel dyes of this invention are also equipped with functionalities which serve in the coupling of these dyes to a variety of biological substrates and other organic molecules, forming conjugates resulting in the fluorescent labeling of the substrates. The novel compounds are intended for use in analytical techniques for the detection and measurement of biological and clinical compounds of interest.
Typical examples of such compounds are bacteria, viruses, enzymes, drugs, blood groups, hormones, environmental contaminents, nucleotides, chemically modified oligo- and polynucleotides, toxins, food, genes and cells.
2) Background Art
Methine and polymethine dyes being in the form of cyanine, merocyanine or squaraine dyes have been known for quite some time and a few are commercially available. None of the previously known dyes, however, appears to have cyano groups in the alpha carbon relative to the nucleus of which they are composed and even fewer possess functionality which serves in the attachment of the polymethine dyes to biological substrates of interest.
Accordingly, one or more of the following objects can be achieved by the practice of this invention. It is an object of this invention to provide novel alpha-cyano-methine and polymethine dyes with interesting physicochemical properties which are very important in the labeling of cells and other biological substrates. Another object for this invention is to provide novel cyano-methine and cyano-polymethine dyes which may be readily coupled to compounds of clinical interest. A further object of the present invention is to provide novel cyano-methine and cyano-polymethine dyes which will exhibit distinct fluorescence excitation and emission spectra, corresponding to that of the specific class of chromophors. A still further object of this invention is to provide processes for the use of derivatives of the dyes for the detection and mesurement of biological compounds. These and other objects will readily become apparent to those skilled in the art in the light of the teachings herein set forth.